ALBERT

Description: Introduction: Proof of principle that adoptively transferred NK cells can mediate regression of hematological malignancies has recently been established in the clinic. Despite recent advances in the field, the overall efficacy of NK-cell based immunotherapy remains limited. Directing cellular migration to tumor-bearing tissues could be used as a method to improve the efficacy of NK cell-based immunotherapy. As most hematological malignancies arise from bone marrow (BM) compartments, we investigated the potential of genetic modification of NK cells to express high levels of the BM homing chemokine receptor CXCR4 to improve NK cell migration to BM compartments in vivo. Methods: Human NK cells were expanded ex vivo in G-rex flasks for 14 days using irradiated EBV-LCL feeder cells and IL-2 containing media. Electroporation (EP) of NK cells with mRNA coding for the wild-type CXCR4 receptor (WT CXCR4) and the gain-of-function mutation CXCR4 receptor (CXCR4-R334X) was performed using the MaxCyte GT instrument. Cell viability and receptor expression was assessed by flow cytometry using a BD LSR II Fortessa. In vitro transwell migration assays towards the CXCR4 ligand SDF-1α were performed in serum-free media over 2 hours at +37¡C. Pretreatment of NK cells with 100 uM of plerixafor for 30 min at +4¡C prior to migration assays was used for CXCR4 blockade experiments. In vivo homing studies were performed with bioluminescence tracking of luciferase-transfected NK cells in NSG mice. Animals were imaged using an IVIS Bioluminescence imager 1 and 24 hours after adoptive NK cell transfer. Results: EP of ex vivo expanded NK cells with either WT CXCR4 or CXCR4-R334X mRNA both resulted in a substantial increase in CXCR4 surface expression for up to 36 hours compared to non-EP NK cell controls. In vitro assays showed CXCR4-R334X transfected NK cells had superior migration to SDF-1α compared to both WT CXCR4 transfected and control NK cells, with an average 40% increase in their migration capacity towards SDF-1α compared to non-transfected NK cells (n=10 donors). This augmented migration capacity was abrogated when the CXCR4 receptor was selectively blocked with plerixafor. To confirm that CXCR4-R334X modified NK cells had improved BM homing capacity in vivo, we compared the distribution of these cells using bioluminescent imaging (BLI) after transfection with luciferase mRNA and intravenous injection into NSG mice (n=3). Twenty-four hours after adoptive transfer, CXCR4-R334X mRNA EP NK cells had improved homing to BM compartments such as the vertebrae, sternum ribs, and femurs compared to their unmodified NK cells counterparts (figure). Conclusions: The data demonstrate that genetic modification of NK cells with CXCR4-R334X mRNA can be utilized to efficiently direct their homing of infused NK cells to BM compartments in vivo. We hypothesize that CXCR4-modified NK cells can be utilized to improve the efficacy of adoptive NK cell immunotherapy for patients with BM-residing malignancies such as leukemia and multiple myeloma. Emily R. Levy is a predoctoral candidate in the Molecular Medicine program of Institute for Biomedical Sciences at the George Washington University. This work is from a dissertation to be presented to the above program in partial fulfillment of the requirements for the Ph.D. degree. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Although the proteasome inhibitor bortezomib has significantly improved the survival of patients with multiple myeloma (MM), most patients treated with this drug eventually develop progressive disease. Recent data indicate that MM cells can evade bortezomib-induced cell death by undergoing autophagy as a consequence of endoplasmatic reticulum (ER)-stress triggered by proteasome inhibition. Here we show that bortezomib-evading MM cells become highly sensitized to killing by natural killer (NK) cells via ER-stress-induced reduction of the NK cell inhibitory molecule HLA-E that is normally expressed at high levels on the surface of MM cells. High-resolution flow cytometry-based assays revealed augmented NK cell recognition and degranulation against bortezomib-exposed MM cells (3 fold higher compared to untreated MM controls) was restricted to NK cells exclusively controlled by the HLA-E-binding inhibitory receptor NKG2A (NKG2ASP NK cells) (Figure 1). In contrast, due to unchanged high expression of other HLA class I molecules on the surface of bortezomib-exposed MM cells there was no augmentation in degranulation by NK cells controlled by other inhibitory HLA class I-binding receptors, such as killer immunoglobulin-like receptors (KIRs). Compared to their non-expanded counterparts, ex vivo expanded NK cells have previously been shown to have an increased proportion of NKG2ASP NK cells (50% vs 25%, p

Description: Introduction : Natural killer (NK) cells are highly cytotoxic immune cells that can kill tumor cells via release of cytotoxic granulae as well as through induction of tumor apoptosis by ligands that bind death receptors expressed on the target cells. Clinical trials have established that adoptive infusions of ex vivo expanded NK cells are safe and can induce tumor regression in selected groups of cancer patients. Recent data suggest that Ewing's sarcoma (EwS), a bone cancer associated with poor survival in the context of metastatic disease, is exquisitely sensitive to killing by NK cells due to low expression of HLA class I molecules that normally prevent NK cell cytotoxicity through interactions with inhibitory NK cell receptors. We and others have recently shown that ex vivo expansion of NK cells causes upregulation of their activation receptors such as NKG2D and death receptor ligands such as TRAIL, which collectively make expanded NK cells more cytotoxic than resting non-expanded NK cells. In an effort to optimize the full therapeutic potential of adoptive NK cell immunotherapy against EwS in the clinic, we investigated the mechanisms utilized by ex vivo expanded NK cells to recognize and kill EwS cells. Methods : Healthy donorNK cells were expanded for 14 days using irradiated EBV-LCL cells in X-Vivo 20 media supplemented with 500 IU/ml IL-2 and 10% AB serum. The EwS cell lines (TC71, RH18X, LG) and the K562 cell line were grown in RPMI media supplemented with 10% FBS. NK cell viability, phenotype, and degranulation were measured by flow cytometry. EwS lysis was measured using 51 Cr release assays. Degradation of perforin to prevent tumor killing via the degranulation pathway was achieved by pre-treating NK cells for 2 hours with 100 nM concanamycin. Blocking antibodies against HLA-A,B,C antigens on EwS cells and against activation receptors on NK cells were added to the respective cells for 30-45 min prior to co-culture. In some experiments, EwS cells were pre-treated with 20 nM bortezomib for 24 hours prior to co-culture with NK cells. Statistical analysis was conducted using the Wilcoxon ranked sum test to determine significance. Results: Ex vivo expanded NK cells were highly cytotoxic against all three EwS cell lines tested, with killing levels comparable to those of the gold-standard NK cell target K562 cells. Suppression of the degranulation pathway using concanamycin revealed a significant reduction in the ability of NK cells to lyse EwS cells (65-71% at baseline vs 10-24% with concanamycin-treated NK cells). Blockade of HLA class I molecules on the EwS cell surface revealed a small but significant increase in NK cell degranulation from 30 to 37%, 32 to 40%, and 20 to 35% against the TC71, RH18X, and LG EwS lines respectively (p

Description: Abstract 3015 Recently, we isolated and expanded a CD8+ T-cell clone from the blood of a patient with regressing renal cell carcinoma following an allogeneic hematopoietic stem cell transplant (HSCT) that killed patient tumor cells in vitro. This CTL clone was found to have tumor specific cytotoxicity, recognizing an HLA-A11-restricted 10-mer peptide named CT-RCC-1. The transcripts encoding this antigen, CT-RCC-8 and CT-RCC-9 were found to be derived from a novel human endogenous retrovirus type E (named CT-RCC HERV-E) located on chromosome 6q. Both transcripts present splice variants that share a common sequence region encoded in the retroviral 5'LTR that is spliced to non-shared regions derived from the protease and polymerase genes, respectively. Remarkably, these transcripts were found to be selectively expressed in the clear cell variant of renal cell carcinoma (ccRCC) with no expression observed in normal tissues or any other type of tumor cell. We have now discovered transcripts encoding the entire envelope gene (env) of the CT-RCC HERV-E provirus are also expressed in ccRCC. Expression of these unique HERV-E env transcripts was detected in kidney cancer cells by RT-PCR, Northern blot, and sequence analysis. The proviral env was found to be expressed concurrent with the previously identified CT-RCC-8 and -9 transcripts, and was only observed in ccRCC cells with no expression observed in any other tumors or normal tissues. We generated 2 peptides derived from the env surface region and 2 peptides derived from the env transmembrane region that were predicted to have a high binding affinity for HLA-A2. Dendritic cells generated from the PBMC of healthy HLA A2+ donors were pulsed with one of these 4 env peptides then were used to stimulate autologous T-cells in vitro. After three stimulations, 3 of these peptides were found to expand CD8+ T-cells that secreted IFN-γ when co-cultured with a HERV-E Env expressing ccRCC tumor cell line transfected to express HLA A2+ but did not secrete IFN-γ against the wild type tumor that was HLA A2 negative. Conclusion: The envelope of the CT-RCC HERV-E provirus is expressed selectively in ccRCC and encodes highly immunogenic antigens that can be targeted by cytotoxic T-cells. Our data suggests antigens derived from this newly discovered HERV-E envelope could represent excellent targets for T-cell based immunotherapy for kidney cancer. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1001 The proteosome inhibitor bortezomib sensitizes tumors in-vitro and in-vivo to autologous NK cell killing by augmenting NK cell TRAIL and perforin/granzyme-mediated caspase-8 activity (Lundqvist et al, Blood 2009). This effect occurs independent of tumor MHC class I expression, suggesting drug-induced tumor sensitization to autologous NK cell killing could be used to override the dominant inhibitory signaling that occurs via KIRs. Based on preclinical data, we initiated a phase I clinical trial to explore the safety and antitumor efficacy of escalating doses of adoptively infused ex-vivo expanded autologous NK cells following bortezomib treatment in patients (pts) with a variety of advanced malignancies refractory to conventional therapy. Pts underwent a 15–20L apheresis to isolate NK cells that were enriched using Miltenyi immuno-magnetic beads to deplete CD3+ T cells followed by CD56+ selection. Enriched NK cells (5–12 × 107cells) were expanded ex-vivo over 14–27 days using an irradiated clinical grade EBV-LCL feeder cell line. On day −3, pts receive a single injection of pentostatin (4mg/m2) to deplete Tregs followed by an injection of bortezomib (1.3 mg/m2) on day −1 to sensitize tumors to NK cell killing. Cohorts 1–4 received a single infusion of ex-vivo expanded NK cells on day 0 in a dose escalating fashion (5×106, 1×107, 5×107, and 1×108 NK cells/kg; 3–6 pts per cohort). Cohorts 5–6 received 1 × 108 NK cells/kg on day 0 and a second escalating dose of NK cells infused on day +5 (5 × 107 and 1 × 108 NK cells/kg respectively) following treatment with a second dose of bortezomib given on day +4. To maintain NK cell viability and TRAIL surface expression, 2 million IU/m2 of IL-2 was given s.c. every 12 hrs on days 0 through +6 in cohorts 1–4 and days 0 through +9 for cohorts 5–6. Pts with stable disease or regression were eligible to receive additional cycles of therapy. Twenty pts received a total of 73 adoptive NK cell infusions. 58/59 (98%) NK cell cultures expanded successfully to achieve the target NK cell dose. NK cells harvested 14–27 days after expansion contained a median 99.7% (range 92–100) CD3-/CD56+ NK cells and had a median 87% (range 71–93) viability. NK cells for the first infusion given on day 0 expanded a median 199 fold (range 58–6647) ex-vivo after a median 14 days of culture (range 14–22). NK cells given on day +5 expanded a median 1298 fold (range 243-20, 196) after a median 20 days of culture (range 19–27). For cohorts 3–4, NK cells peaked in circulation at a median 382cells/μL (range 60–1851) at median 7 days following adoptive transfer. For cohorts 5–6, NK cells increased in the circulation a median 6.0 fold (range 1.4–7.0) over baseline, peaking at a median 266 cells/μL (range 61–301) at a median 10 days following adoptive transfer. No grade II–IV toxicities related to NK cell transfer were observed. The most common adverse events were attributed to IL-2 therapy including grades I-II fever, renal insufficiency, edema and hypotension. Four pts developed elevated free T4 levels and low TSH levels following NK cell therapy consistent with acute thyroiditis; two became hypothyroid and required thyroid replacement therapy. Best clinical response to date in the first 20 pts treated included 6 pts with progressive disease, 10 pts with stable disease (including 2 pts with metastatic tumors who had more than a 30% decline in serum tumor markers) and 4 pts with a minor response (2 pts with renal cell carcinoma (RCC) and 2 pts with chronic lymphocytic leukemia (CLL)). Thirteen of 20 pts (66%) went on to receive more than 1 NK cell infusion including 1 pt who received 6 cycles, 3 pts who received 5 cycles, 4 pts who received 4 cycles, 3 pts who received 3 cycles and 1 pt who received 2 cycles before going off study for either progressive disease or personal preference. In conclusion, this study has established that 2 infusions of ex-vivo expanded autologous NK cells at a dose of 1 × 108 cells/kg given on days 0 and +5 are safe with preliminary evidence for antitumor immunity being observed against metastatic RCC and treatment refractory CLL. With the exception of thyroiditis, infusions of ex-vivo expanded NK cells were well tolerated with no grade III/IV toxicities observed to date. This phase I study continues to accrue pts with cohorts 7–10 intended to establish the maximum tolerated dose of ex-vivo expanded NK cells that can be infused on day 5 (up to a dose of 1 × 109 NK cells/kg). Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4355 Natural killer (NK) cells comprise a critical component of the innate immune system that controls infections and tumor transformation. Although proof of principle supporting the antitumor effects of adoptively infused autologous and allogeneic NK cells has been established, overall response rates appear low, possibly related to their inability to traffic to lymphoid tissues and the bone marrow where hematological malignancies such as AML, lymphomas, and multiple myeloma reside. CD62L receptors play a critical role in the recruitment of NK cells from the circulation to the bone marrow and lymph nodes. Recently, investigators have developed a number of approaches to expand NK cells ex vivo for adoptive infusion in patients with cancer. Although NK cells cultured with irradiated EBV transformed lymphoblastoid cell lines (EBV-LCL) can be expanded by more than 1000 fold and are more cytotoxic to tumor cells compared to resting or IL-2 activated NK cells, these expanded cells undergo a substantial reduction in CD62L surface expression which could hinder their ability to be recruited into the tumor microenvironment. NAM, a specific inhibitor of NAD (+) dependent enzymes, was shown to up-regulate surface expression of CD62L on freshly isolated NK cells cultured in feeder cell-free cytokine-containing media. Based on this observation, we hypothesized that CD62L expression would be increased on NK cells expanded ex vivo using EBV-LCL feeders by adding NAM to the culture media. Methods: Human NK cells were isolated from PBMCs of 5 healthy volunteers by depleting CD3+ T cells and subsequently selecting CD56+ cells using immuno-magnetic beads. NK cells were expanded ex vivo in flasks over 21 days by co-culturing with an irradiated EBV-LCL feeder cell line at 20:1 LCL to NK cell ratio in media containing 500 IU/mL of IL-2. NAM was added to the media on culture day 7 at a 5 mM or 7.5 mM concentration and expanded NK cells were evaluated every 2–3 days by FACS to assess their phenotype and viability and by cytotoxicity assays to assess their cytotoxicity against K562 and renal cell carcinoma targets. The effects of NAM on the proliferative capacity of ex vivo expanded NK cultures were likewise assessed. Results: NK cell expansion cultures containing NAM had substantially higher CD62L surface expression compared to cultures without NAM, with this effect being most pronounced in NK cell cultures containing the higher concentration (7.5 mM) of NAM (see figure). On NK cell culture days 12, 14, 16, and 21, the surface MFI of CD62L was a median 1.62, 2.44, 2.03 and 3.65 fold higher on NK cells cultured in 7.5 mM of NAM compared to cultures without NAM (p =0.017, 0.012, 0.037 and 0.046 respectively; paired t-test). No significant difference in surface expression of NK cell TRAIL, NKG2D, NKG2A, NKp30, NKp46, KIR2DL1, KIR3DL1, KIR2DL2/DL3, CD44, CD95, and CD200R was observed between NAM containing cultures and controls although NKp44 expression was slightly higher in cultures that did not contain NAM. NAM-treated and untreated NK cells had similar cytotoxic function against K562 and renal cell carcinoma (RCC) tumor cell lines. The viability of NK cells, assessed by Trypan Blue and Annexin V staining, was unaffected by NAM. The addition of NAM to cell culture media did result in a dose-dependent reduction in ex vivo NK cell expansion: on days 14 and 16, NK cells expanded a median 926 and 2963 fold in control media compared to a median 443 and 1151 fold in media containing 5 mM NAM (p=0.0029 and 0.0074 respectively) and a median 359 and 732 fold in media containing 7.5 mM NAM (p=0.0019 and 0.0003 respectively). Although the proliferation of NK cells was reduced, ex vivo expansion of NK cells still occurred in NAM containing media with NK cells expanding a median 2785 and 1360 fold by day 18 in cultures containing 5 and 7.5 mM of NAM, respectively. Conclusion: The addition of NAM to NK cell expansion cultures substantially increased surface expression of CD62L on NK cells without any deleterious effect on their cytotoxic function. Although NAM reduced ex vivo NK cell proliferation, NK cells still expanded more than 1000 fold in NAM containing media. These data suggest NAM-induced increases in CD62L surface expression could be used as a novel method to improve the homing capacity of ex vivo expanded NK cells to the bone marrow and the lymphoid organs, potentially enhancing their antitumor effects when adoptively infused in patients with hematological malignancies. Disclosures: Peled: Gamida Cell Ltd. Cell Therapy Technologies: Employment. Frei:Gamida Cell Ltd. Cell Therapy Technologies: Employment.

Description: Background Bronchiolitis Obliterans Syndrome (BOS) is a late-onset non-infectious pulmonary complication of HSCT, resulting in obstructive lung disease. BOS is thought to be a manifestation of chronic graft versus host disease (cGVHD). BOS can also occur after lung transplantation, where it is believed to represent chronic rejection of the lung allograft. In both conditions, the mainstay of therapy includes augmentation of systemic immunosuppression. However, this approach has limited efficacy and is associated with deleterious consequences including an increased risk of infection and decreased graft versus tumor/leukemia effects. We investigated whether targeted, local delivery of inhaled cyclosporine could improve or stabilize lung function in BOS patients. Methods HSCT recipients with BOS, ages 10-80, were eligible if they met the following inclusion criteria: FEV110% compared to pre-transplant FEV1, no evidence of pulmonary infection as a causative etiology, and one of the following: FEV1/FVC ratio

Description: Introduction: Umbilical cord blood (UCB) grafts are the only option for a significant minority of patients who require hematopoietic stem cell transplantation (HSCT) but lack a suitable related or unrelated donor. While UCB can serve as a suitable 'off the shelf' graft for many patients, units with the best HLA match often contain low and sometimes insufficient numbers of CD34+ cells for use in transplantation, particularly for adult patients. Furthermore, UCB grafts contain lower CD34+ cells number compared to BM or PBSC grafts, which leads to longer engraftment times and a higher risk of graft failure. BM and PBSC grafts are usually injected intravenously, homing to the bone marrow after several hours in the circulation. During this time, CD34+ cells are lost in the lungs, liver and spleen with typically 〈 20% making it to the bone marrow.1 Investigators have sought to overcome the limitation of low cell dose in UCB grafts and loss of CD34+ cells in the circulation by injecting CD34+ cells directly into the bone marrow space. However, we have recently shown that conventional intrabone delivery methods used in investigational trials to transplant UCB, result in low-level retention of hematopoietic progenitor cells in the intrabone space. Recently, we developed an optimized intrabone (OIB) transplant method using computer controlled low pressure and low volume injection (controlled infusion rate

Description: Abstract 654 Unrelated cord blood (UCB) transplantation is a useful alternative for patients with hematological malignancies or non-malignant hematological disorders lacking an HLA matched donor. However, outcomes for patients with severe aplastic anemia (SAA) undergoing either a single or dual UCB transplant have been disappointing. A recent EBMT/ Eurocord study reported engraftment and 3 year survival rates of only 51% and 38% respectively (Perrault de Latour, Biol Blood Marrow Transplant 2011). We investigated whether co-infusion of a single UCB unit with CD34+ selected cells from a haploidentical relative following a highly immunosuppressive conditioning regimen could improve transplant outcome for patients with SAA refractory to immunosuppressive therapy that lack an HLA matched donor. Subjects with SAA and life-threatening neutropenia (ANC 500 by day 42, 7 of 8 achieving a UCB-derived ANC 〉500 cells/μl. The median time to neutrophil recovery was 10 days (range 10–18 days). One patient failed to engraft with the cord unit, but has had sustained engraftment from the haploidentical donor, and is transfusion independent with a normal neutrophil count 〉25 months post transplant. Acute GVHD grade II developed in 2 patients and one developed limited chronic GVHD. Early T-cell engraftment was predominantly UCB in 7 cases; on day 21, T-cell chimerism was a median 100% cord in origin (range 0–100%). In contrast, myeloid chimerism at engraftment was predominantly haplo-donor in origin and showed 3 phases of engraftment: 1) early myeloid engraftment from the haplo-CD34+ cell donor 2) delayed myeloid engraftment from the cord unit resulting in dual myeloid chimerism and 3) disappearance of the haplo-donor cells with transition towards full cord donor myeloid chimerism (see figure). Mixed lymphocyte reactivity assays performed on post transplant PBMCs showed increasing alloreactivity of cord blood T-cells against the haploidentical donor during the period when myeloid chimerism transitioned towards cord, indicating that the disappearance of haplo-donor myeloid cells occurred as a consequence of rejection by engrafting cord blood T-cells. At a median follow-up of 9 months (range 75 days to 3 years), 7 patients survive, and all are transfusion–independent. One patient died 14 months after transplantation from complications related to CMV pneumonitis. In conclusion, transplantation of haploidentical CD34+ cells can shorten the time to neutrophil recovery in SAA pts undergoing a single UCB transplant. Furthermore, durable full engraftment from donor haploidentical CD34+ cells can occur in the context of cord graft failure. These data suggest co-infusion of allogeneic cord blood with haploidentical CD34+ cells can improve the outcome of UCB transplantation for SAA. Disclosures: Wilder: NCI: Funded in part by NCI contract No. HHSN261200800001E.